ddPCR assay design

Question 1

What is the ideal amplicon length?

Answer 1

60-200 bp

Question 2

What is the ideal amplicon GC content?

Answer 2

40-60%

Question 3

Considerations for primer design.

Answer 3

Length: 15-30 bp
GC content: 50-60%
Tm: 50-65 C
Avoid secondary structure and complementarity at 3’ ends; use G/C at 3’ end, but avoid long stretches of G/C

Question 4

How do we recommend calculating Tm?

Answer 4

Oligocalc tool from Northwestern University, use [salt]=50 mM and [primers]=300 nM

Question 5

Considerations for probe design.

Answer 5

Length: <30 bp
GC content: 30-80% (anneal to strand with more Gs so that probe has more Cs)
Tm: 3-10 C higher than primers
Location: between primers, cannot overlap with primers, but can sit directly next to them)
Avoid 5’ G (quenches fluorescence)

Question 6

What is the recommended quencher?

Answer 6

Black Hole quenchers or other non-fluorescent (dark) quenchers are recommended; TAMRA causes problems.

Question 7

Additional design consideration for SNP and rare mutation detection probes.

Answer 7

Tm enhancers (e.g. minor groove binder probe) are recommended for mutation descrimintation because it allows shorter probes (better descrimination) with higher Tm.

Question 8

What are MGB probes?

Answer 8

Minor groove binder probes - contain an MGB moiety (e.g. DPI3) at the 3’ end.

Question 9

What is a recommended tool for primer and probe design? What changes are required to default settings when using this tool for ddPCR?

Answer 9

Primer3Plus (form MIT)
There are instructions in the application guide.
General settings changes: [divalent cations]=3.8 mM, [dNTPs]=0.8 mM, select correct “Mispriming/Repeat Library”
Advanced settings changes: Table of thermodynamic parameters and salt correction formula should both be set to SantaLucia 1998
Internal Oligo settings: min length 15, Tm 64-70 (65 ideal)

Question 10

After using Primer3Plus, what additional things need to be checked

Answer 10

1. BLAST to check for off-target amplicons
2. check no common SNPs are in the primers or probes
3. check amplicon secondary structure for structure within primer/probe binding sites (using Mfold program)

Question 11

How are gene editing events detected?

Answer 11

Drop off (DOF) experiment: Two probes that bind the amplicon, one over a reference area, one over the cut site. The wild-type will have both colours, but where gene editing events occur, the cut-site probe will drop off, and there will be only a single colour fluorescence..

Question 12

How does the RED algorithm differ to ABS/DQ?

Answer 12

RED has a stricter droplet gating, resulting in fewer droplets being accepted. This algorithm reduces false positives, and is more appropriate to low positive numbers. It reduces the risk of accidentally scoring a false positive for a droplet that may have undergone coalescence/ shearing. Selecting RED after the fact will re-process the data using this algorithm.

Question 13

What is the limit of detection for rare mutation detection by ddPCR, and how does this compare to qPCR?

Answer 13

Around 0.01% (compared to 1% in qPCR) - but can increase number of wells to increase LOD.